GMP-compliant generation, expansion, and a preliminary safety evaluation of CMV-specific t cells using a closed system for post-transplant viral control

Abstract

Abstract Despite advances in transplantation, recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT) remain at high risk for opportunistic viral infections. Cytomegalovirus (CMV) is a leading cause of post-transplant morbidity and mortality. Virus-specific T cell (VST) therapy has emerged as a promising strategy to control CMV infection without increasing the risk of de novo graft-versus-host disease (GVHD). We developed a Good Manufacturing Practices (GMP)-compliant protocol, ImmuneCellVir-I, for generating CMV-specific T cells from the Leukoreduction System (LRS) chamber—a sterile component of the platelet apheresis kit known for its high T cell content. T cells were expanded ex vivo using a cytokine cocktail (IL-2 and IL-7) to promote proliferation and an effector memory phenotype. The manufacturing process was conducted in a closed, automated system, ensuring sterility, reproducibility, and high purity. Specificity was validated in vitro via IFN-γ secretion against CMV-infected targets (AD169 strain) and lack of reactivity against uninfected, partially HLA-matched cells. In vivo testing in NOD scid mice confirmed product persistence, biodistribution, and lack of toxicity. Following ANVISA's approval for a phase I clinical trial, a 45-year-old male patient with refractory classical Hodgkin lymphoma was treated with ImmuneCellVir-I. His prior treatments included ABVD, DHAP, ICE, GVD, brentuximab vedotin (BV), autologous hematopoietic stem cell transplantation, and pembrolizumab, which led to complete remission. In August 2024, he underwent a haploidentical allogeneic HSCT. The post-transplant course was complicated by chronic graft-versus-host disease (overlap subtype), presenting with grade 3 skin and grade 2 oral involvement, which was managed with prednisone and tacrolimus. In early 2025, the patient developed CMV reactivation with a high viral load (>200,000 copies/mL) that proved refractory to ganciclovir, which was discontinued due to hematologic toxicity and the detection of viral resistance associated with an M460V mutation. The first infusion of ImmuneCellVir-I occurred on day 0 and did not result in immediate viral control; viremia peaked at >500,000 copies/mL on day 14, accompanied by transient leukocytosis. Viral decline began on day 21. A second infusion on day 36 was followed by a rapid drop in viral load and emergence of a CMV-specific T cell response detected by ELISPOT on day 37, which intensified over the following two weeks. No GVHD flare or CRS was observed following CTL infusions. A transient oral lesion flare occurred on day 21, coinciding with immunosuppressive taper, and was classified as limited. This case illustrates the feasibility, safety, and antiviral efficacy of ImmuneCellVir-I in the treatment of drug-refractory CMV infection after haploidentical HSCT, even in the context of active chronic GVHD. These findings support further investigation of CMV-specific T cell therapy as a valuable adjunct in managing opportunistic infections in immunocompromised hosts. Funding: Ministry of Health of Brazil, SIPAR (NUP) 25000.156425/2023-06